Methods of modifying myocardial infarction expansion

ABSTRACT

A bioscaffolding can be formed within a post-myocardial infarct region sufficient to cause attenuation of a rate of myocardial infarct expansion. A bioscaffolding may further be formed in the post-myocardial infarct region to cause an increase in posterior left ventricular wall thickness. The gel or bioscaffolding can be formed from a mixture of gel components of different gelation systems. For example, a bioscaffolding can be formed by mixing at least two different components of at least two different two-component gelation systems to form a first mixture and by mixing at least two different components (other than the components that make up the first mixture) of the at least two different two-component gelation systems to form a second mixture.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 60/855,593 filed on Jan. 18, 2007, which is incorporatedherein by reference in its entirety.

FIELD

Post-myocardial infarction treatments and compositions.

BACKGROUND

Ischemic heart disease typically results from an imbalance between themyocardial blood flow and the metabolic demand of the myocardium.Progressive atherosclerosis with increasing occlusion of coronaryarteries leads to a reduction in coronary blood flow. “Atherosclerosis”is a type of arteriosclerosis in which cells including smooth musclecells and macrophages, fatty substances, cholesterol, cellular wasteproduct, calcium and fibrin build up in the inner lining of a bodyvessel. “Arteriosclerosis” refers to the thickening and hardening ofarteries. Blood flow can be further decreased by additional events suchas changes in circulation that lead to hypoperfusion, vasospasm orthrombosis.

Myocardial infarction (MI) is one form of heart disease that oftenresults from the sudden lack of supply of oxygen and other nutrients.The lack of blood supply is a result of a closure of the coronary artery(or any other artery feeding the heart) which nourishes a particularpart of the heart muscle. The cause of this event is generallyattributed to arteriosclerosis in coronary vessels.

Formerly, it was believed that an MI was caused from a slow progressionof closure from, for example, 95% then to 100%. However, an MI can alsobe a result of minor blockages where, for example, there is a rupture ofthe cholesterol plaque resulting in blood clotting within the artery.Thus, the flow of blood is blocked and downstream cellular damageoccurs. This damage can cause irregular rhythms that can be fatal, eventhough the remaining muscle is strong enough to pump a sufficient amountof blood. As a result of this insult to the heart tissue, scar tissuetends to naturally form.

Various procedures, including mechanical and therapeutic agentapplication procedures, are known for reopening blocked arties. Anexample of a mechanical procedure includes balloon angioplasty withstenting, while an example of a therapeutic agent application includesthe administration of a thrombolytic agent, such as urokinase. Suchprocedures do not, however, treat actual tissue damage to the heart.Other systemic drugs, such as ACE-inhibitors and Beta-blockers, may beeffective in reducing cardiac load post-MI, although a significantportion of the population that experiences a major MI ultimately developheart failure.

An important component in the progression to heart failure is remodelingof the heart due to mismatched mechanical forces between the infarctedregion and the healthy tissue resulting in uneven stress and straindistribution in the left ventricle (LV). Once an MI occurs, remodelingof the heart begins. The principle components of the remodeling eventinclude myocyte death, edema and inflammation, followed by fibroblastinfiltration and collagen deposition, and finally scar formation fromextra-cellular matrix (ECM) deposition. The principle component of thescar is collagen which is non-contractile and causes strain on the heartwith each beat. Non-contractility causes poor pump performance as seenby low ejection fraction (EF) and akinetic or diskinetic local wallmotion. Low EF leads to high residual blood volume in the ventricle,causes additional wall stress and leads to eventual infarct expansionvia scar stretching and thinning and border-zone cell apoptosis. Inaddition, the remote-zone thickens as a result of higher stress whichimpairs systolic pumping while the infarct region experiencessignificant thinning because mature myocytes of an adult are notregenerated. Myocyte loss is a major etiologic factor of wall thinningand chamber dilation that may ultimately lead to progression of cardiacmyopathy. In other areas, remote regions experience hypertrophy(thickening) resulting in an overall enlargement of the left ventricle.This is the end result of the remodeling cascade. These changes alsocorrelate with physiological changes that result in increase in bloodpressure and worsening systolic and diastolic performance.

SUMMARY OF INVENTION

A bioscaffolding can be formed within a post-myocardial infarct regionsufficient to cause attenuation of a rate of myocardial infarctexpansion. The bioscaffolding can be formed within the post-myocardialinfarct region by combining components of two-component systems.

The bioscaffolding can be formed from a mixture of gel components ofdifferent two-component gelation systems. In some embodiments, abioscaffolding can be formed by mixing at least two different components(which do not gel upon mixing) of at least two different two-componentgelation systems to form a first mixture and by mixing at least twodifferent components (other than the components that make up the firstmixture and which do not gel upon mixing) of the at least two differenttwo-component gelation systems to form a second mixture. A treatmentagent, such as a cell type or a growth factor, can be added to eitherthe first mixture or the second mixture. The first mixture can then beco-injected with the second mixture to form a bioscaffolding in aninfarct region for treatment thereof. The first and second mixtures canbe co-injected with a dual-lumen delivery device, which can include, butare not limited to, a dual syringe, a dual-needle left-ventricleinjection device, a dual-needle transvascular wall injection device andthe like.

In some embodiments, a bioscaffolding can be formed by mixing at leasttwo different gelation components (which do not gel upon mixing) to forma first mixture. A treatment agent, such as a cell type or a growthfactor, can be added to the first mixture. The first mixture can then beco-injected with a second gelation component to form a bioscaffolding onor within an infarct region for treatment thereof. The first mixture andthe gelation component can be co-injected with, a dual-lumen deliverydevice, which can include, but are not limited to, a dual syringe, adual-needle left-ventricle injection device, a dual-needle transvascularwall injection device and the like.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B illustrate the progression of heart damage once the build-upof plaque in an artery induces an infarct to occur.

FIGS. 2A-2B illustrate left ventricular posterior wall thicknesses forexperimental groups.

FIGS. 3A-3B illustrate left ventricular end-diastolic volumes forexperimental groups.

FIGS. 4A-4B illustrate relative changes in left ventricular ejectionfraction for experimental groups.

FIGS. 5A-5C illustrate serial changes in radio-opaque markers positionedwithin myocardial infarct regions.

FIG. 6A illustrates left ventricular volume for experimental groups.

FIG. 6B illustrates left ventricular ejection fraction for experimentalgroups.

FIG. 7A illustrate left ventricular wall stress for remote regions ofexperimental groups.

FIG. 7B illustrates left ventricular wall stress for myocardial infarctregions.

FIGS. 8A-8B illustrate interstitial matrix metallopeptidase (MMP)activity for experimental groups.

FIG. 9 illustrates myocardial blood flow to tissue regions of theexperimental groups.

FIGS. 10A-10B illustrate percentage of myocardial infarction regions forexperimental groups.

FIG. 11 illustrates regional myocardial stiffness for experimentalgroups.

FIG. 12 illustrates histological tissue sections stained for hematoxylinand eosin for experimental groups.

FIG. 13 illustrates photomicrographs for Alcian blue sections counterstained with nuclear fast red.

FIG. 14A illustrates histochemical staining for neutrophils.

FIG. 14B illustrates immunohistochemical staining for macrophages.

FIGS. 15A-15B illustrate left ventricular myocardial morphometricmeasurements of percent collagen within each left ventricular region forexperimental groups.

FIG. 16 illustrates capillary density in full thickness left ventricularsections for experimental groups.

FIG. 17A illustrates levels of soluble collagen for experimental groups.

FIG. 17B illustrates levels of hydroxyproline for experimental groups.

FIG. 18A illustrates left ventricular MMP profiles of MMP-9 forexperimental groups.

FIG. 18B illustrates left ventricular MMP profiles of MMP-2 forexperimental groups.

FIGS. 19A-19B illustrate MMP-2 profiles for experimental groups.

FIG. 20 illustrates an embodiment of a dual bore delivery device.

FIGS. 21A-21B illustrate an alternative embodiment of a dual boredelivery device.

FIGS. 22A-22C illustrate a second alternative embodiment of a dual boredelivery device.

DETAILED DESCRIPTION

FIGS. 1A-1B illustrate the progression of heart damage once the build-upof plaque induces an infarct to occur. FIG. 1A illustrates a site 10where blockage and restricted blood flow can occur from, for example, athrombus or embolus. FIG. 1B illustrates resultant damage area 20 to theleft ventricle that can result from the lack of oxygen and nutrient flowcarried by the blood to the inferior region left of the heart. Thedamage area 20 will likely undergo remodeling, and eventually scarring,resulting in a non-functional area.

A bioscaffolding formed of two components and applied in situ to theleft heart ventricle can be used to treat post-myocardial infarctiontissue damage. In one embodiment, the bioscaffolding is a gel formedfrom a gelation system. “Bioscaffolding” and “two-component gelationsystem” and “two-component gel system” and “gelation system” and“composite material” are hereinafter used interchangeably. Examples oftwo-component gelation systems include, but are not limited to, alginateconstruct systems, fibrin glues and fibrin glue-like systems,self-assembled peptides and combinations thereof. Each component of thetwo-component gelation system may be co-injected to an infarct region bya dual-lumen delivery device. Examples of dual-lumen delivery devicesinclude, but are not limited to, a dual syringe, dual-needleleft-ventricle injection devices, dual-needle transvascular wallinjection devices and the like.

In some embodiments, at least one cell type may be co-injected with atleast one component of the two-component gelation system to an infarctregion. In some embodiments, the cells may be mixed with at least onecomponent of the two-component gelation system before the two-componentsare co-injected to the infarct region. Examples of cell types, include,but are not limited to, localized cardiac progenitor cells, mesenchymalstem cells, bone marrow derived mononuclear cells, adipose stem cells,embryonic stem cells, umbilical cord blood derived stem cells, smoothmuscle cells or skeletal myoblasts.

In some applications, the two-component gelation system includes afibrin glue. Fibrin glue consists of two main components, fibrinogen andthrombin. Fibrinogen is a plasma glycoprotein of about 340 kiloDaltons(kDa) in its endogenous state. Fibrinogen is a symmetrical dimercomprised of six paired polypeptide chains, alpha, beta and gammachains. On the alpha and beta chains, there is a small peptide sequencecalled a fibrinopeptide which prevents fibrinogen from spontaneouslyforming polymers with itself. In some embodiments, fibrinogen ismodified with proteins. Thrombin is a coagulation protein. When combinedin equal volumes, thrombin converts the fibrinogen to fibrin byenzymatic action at a rate determined by the concentration of thrombin.The result is a biocompatible gel which gelates when combined at theinfarct region. Fibrin glue can undergo gelation between about 5 toabout 60 seconds. Examples of other fibrin glue-like systems include,but are not limited to, Tisseel™ (Baxter), Beriplast P™ (AventisBehring), Biocol® (LFB, France), Crosseal™ (Omrix Biopharmaceuticals,Ltd.), Hemaseel HMN® (Haemacure Corp.), Bolheal (Kaketsuken Pharma,Japan) and CoStasis® (Angiotech Pharmaceuticals).

In some applications, the two-component gelation system includesself-assembled peptides. Self-assembled peptides generally includerepeat sequences of alternating hydrophobic and hydrophilic amino acidchains. The hydrophilic amino acids are generally charge-bearing and canbe anionic, cationic or both. Examples of cationic amino acids arelysine and arginine. Examples of anionic amino acids are aspartic acidand glutamic acid. Examples of hydrophobic amino acids are alanine,valine, leucine, isoleucine or phenylalanine. Self-assembled peptidescan range from 8 to 40 amino acids in length and can assemble intonanoscale fibers under conditions of physiological pH and osmolarity. Insufficient concentration and over time, the fibers can assemble into aninterconnected structure that appears macroscopically as a gel.Self-assembled peptides typically undergo gelation between severalminutes to several hours. Examples of self-assembled peptides include,but are not limited to: AcN-RARADADARARADADA-CNH₂ (RAD 16-II) wherein Ris arginine, A is alanine, D is aspartic acid, and Ac indicatesacetylation; VKVKVKVKV-PP-TKVKVKVKV-NH₂ (MAX-1) wherein V is valine, Kis lysine and P is proline; and AcN-AEAEAKAKAEAEAKAK-CNH₂ wherein A isalanine, K is lysine and E is glutamic acid (EAK 16-II). Self-assembledpeptides show good cytocompatibility, as represented by cell adhesion,cell migration and proliferation.

In some applications, the two-component gelation system is an alginateconstruct system. One component may be an alginate conjugate (oralginate alone) which can include alginate and a protein constituent.The second component may be a salt. Examples of alginate conjugates caninclude, but are not limited to, alginate-collagen, alginate-laminin,alginate-elastin, alginate-collagen-laminin and alginate-hyaluronic acidin which the collagen, laminin, elastin, collagen-laminin or hyaluronicacid is covalently bonded (or not bonded) to alginate. Examples of saltswhich can be used to gel the alginate constructs include, but are notlimited to, calcium chloride (CaCl₂), barium chloride (BaCl₂) orstrontium chloride (SrCl₂). When the components are combined, forexample, alginate-collagen and calcium chloride, the resulting gel has astorage modulus of approximately 1 kiloPascal.

In one embodiment, the alginate construct is alginate-gelatin. Themolecular weight of the gelatin may be in the approximate range of 5 kDato 100 kDa. The relatively low molecular weight of gelatin offersprocessing advantages in that it is more soluble and has lower viscositythan hydrogels of higher molecular weight. Another advantage of gelatinis that it contains from 1 to 4 RGD (arginine-glycine-aspartic acidpeptide sequence) sites per molecule. RGD is a common cell adhesionligand and would increase the retention of cells within the infarct zonewhere the bioscaffolding is formed. The cells retained by the RGD sitesmay be cells co-injected with the bioscaffolding components or dispersedthroughout a component of the system.

The gelatin may be a porcine gelatin or a recombinant human gelatin. Theporcine gelatin is a hydrolyzed type 1 collagen extracted from porcineskin. In one embodiment, the molecular weight of the porcine gelatin isapproximately 20 kDa. The human gelatin is produced by bacteria usinghuman genetic material. The human recombinant gelatin is equivalent tothe porcine gelatin but may reduce the likelihood of an immune responsewhen injected into an infarct region of a human subject.

Alginate is a linear polysaccharide derived from seaweed and containsmannuronic (M) and guluronic acid (G), presented in both alternatingblocks and alternating individual residues. It is possible to use someof the carboxyl groups of the alginate as sites to graft useful celladhesion ligands, such as collagen, laminin, elastin and other peptidefragments of the ECM matrix, forming an alginate conjugate, becausealginate does not have RGD groups for cell retention.

An alginate-gelatin conjugate is valuable because it combinescharacteristics of alginate with characteristics of gelatin, whichinclude, but are not limited to, RGD sites and immunocompatibility.Characteristics of alginate include rapid, almost instantaneousgelation, and an immune stimulating effect. The alginate-gelatinconjugate can be formed of approximately 1% to 30% and more particularlyapproximately 10% to 20% gelatin (either porcine or human recombinant)and approximately 80% to 90% alginate. The relatively lower proportionof alginate-gelatin conjugate is used to retain gelation capacity oncecombined with pure alginate because the alginate carboxyl groups ofalginate that cause the gelation may be bound up in the alginate-gelatinconjugate.

Two-component gelation systems exhibit different characteristicsrelative to one another including, but not limited to, pore size,storage modulus and gelation time. The gelation system behaves as asieving media and therefore includes small pores. “Pore size” refers tosmall, vacuous openings within the gel. “Storage modulus” refers to thestrength or the stiffness of the material upon gelation. Storage moduluscan be measured by a rheometric instrument. “Gelation time” refers tothe kinetics of gelation, the decrease in viscous modulus. Alginateconstructs can gel within about 1 second, while fibrin glues can gelbetween about 5 seconds and about 60 seconds. Self-assembled peptidestypically undergo gelation between several minutes to several hours.

In embodiments in which cells are co-injected with the two-componentgelation system, or mixed with one component before combining the twocomponents, the gelation system can exhibit different characteristicsrelative to one another relating to the cells. Such characteristics caninclude, but are not limited to, morphology of the cells, cellsurvivability, encapsulation efficiency and/or cell concentration.“Morphology” refers to the physical structure of the cells. In the caseof hMSC, the natural morphology is a flattened spindle-shapedmorphology. “Cell survivability” is the amount of time that the cellsremain viable within the gel post-injection. “Encapsulation efficiency”refers to the fraction of the initial number of cells in suspension thatare entrapped within the gel. “Cell concentration” is the encapsulationefficiency divided by the volume of gel formed.

A characteristic which affects the encapsulation efficiency is thedifference in viscosity (η) of the two components. If the difference inviscosity between the two components of the gelation system is large,then the encapsulation efficiency is high only when cells are in thehigh viscosity component. However, if the viscosity of each individualcomponent is lowered without compromising the gelation kinetics, theencapsulation efficiency increases dramatically. For a catheter-baseddelivery system, low viscosity components are very helpful forsuccessful delivery. A successful application of the two components(which are in solution before delivery) can be dependent upon lowviscosity of the individual components.

Modified Gelation Systems

In some embodiments, a bioscaffolding can be formed from a mixture ofgel components of different gelation systems. For example, abioscaffolding can be formed by mixing at least two different components(which do not gel upon mixing under standard cath lab processconditions) of at least two different two-component gelation systems toform a first mixture, and, by mixing at least two different components(other than the components that make up the first mixture and which donot gel upon mixing under standard cath lab process conditions) of theat least two different two-component gelation systems to form a secondmixture. “Gel” generally refers to a semirigid colloidal material formedupon the combination of two different components or two differentmixtures. A treatment agent, such as a cell type or a growth factor, canbe added to either the first mixture or the second mixture. The firstmixture can then be co-injected with the second mixture to form abioscaffolding in an infarct region for treatment thereof In someembodiments, a bioscaffolding can be formed by mixing at least twodifferent gelation components (which do not gel upon mixing understandard cath lab process conditions) to form a first mixture. Atreatment agent, such as a cell type or a growth factor, can be added tothe first mixture. The first mixture can then be co-injected with agelation component to form a bioscaffolding on an infarct region fortreatment thereof. In some embodiments, the treatment agent can beco-injected with the first mixture or the gelation component withoutfirst interdispersing the treatment agent within the first mixture orthe gelation component.

In some embodiments, an alginate construct system can include analginate-gelatin solution as a first component and a calcium chloridesolution as a second component. In some embodiments, human mesenchymalstems cells (hMSC) are suspended in one component of the gelationsystem. hMSC are thought to be capable of both self renewal anddifferentiation into bone, cartilage, muscle, tendon and fat. hMSC alsogive rise to a variety of mature cell types via a step-wise maturationprocess called mesengenesis. The natural morphology of hMSC is elongatedand spindle shaped. The gelatin provides RGD sites for cellular adhesioni.e. adhesion of hMSC. Alginate construct systems exhibit rapid gellingkinetics. When combined, alginate-gelatin and calcium chloride gel toform a bioscaffolding in less than 1 second. The resulting gel has astorage modulus of approximately 1 kiloPascal. In application, cellsurvivability has been observed up to at least 12 days. Encapsulationefficiency is approximately 99%. However, the small pore size ofalginate construct systems, which is from about 2 nm to about 500 nm,can lead to low cell spreadability as observed by the round morphologyof the hMSC cells over time. “Cell spreading” refers to the naturallyoccurring morphology of cells. Advantages of alginate construct systemsinclude, but are not limited to, enhanced immune response (a controlledforeign body response) to effect positive remodeling of the injuredmyocardium, and immunoprotectivity, by shielding via its small poresize, the encapsulated cells from this enhanced immune response(protected from host immune response), instantaneous gelation kinetics,substantial or complete non-adherence to a needle when injected, andlong term cell viability. Furthermore, alginate construct systemsdegrade slowly (at least 8 weeks in vivo).

Fibrin glue can include fibrinogen (modified or not modified withprotein constituents) as a first component and thrombin as a secondcomponent. In some embodiments, human mesenchymal stems cells (hMSC) aresuspended in one component of the gelation system. Fibrin glue systemsexhibit fast gelling kinetics, but not as rapid as alginate constructsystems. When combined, fibrinogen and thrombin gel form abioscaffolding in about 5 seconds to about 10 seconds. The resulting gelhas a storage modulus of approximately 3 kiloPascals which is higherthan that of alginate construct systems. A higher storage modulus mayimprove mechanical reinforcement at the infarct region. In application,cell survivability has been observed up to at least 12 days. The poresize of fibrin glue systems is from about 1.5 microns to about 2.5microns and can lead to high cell spreadability of hMSC cells. That is,hMSC cells have been observed to have an elongate and stellatemorphology which is more natural to their endogenous state when comparedto the morphology observed in alginate construct systems alone.Advantages of fibrin glue include, but are not limited to, materialstrength, promotion of angiogenesis, good cytocompatibility (compatiblewith cell growth), good cell morphology (elongated and stellate) andhigh cell proliferation in fibrinogen. One further characteristic offibrin based gels is that they degrade within 2 weeks in vivo.

In some embodiments, a bioscaffolding is formed from mixing componentsof at least two gelation systems. For example, a first component of afirst two-component gel and a first component of a second two-componentgel can be combined to form a first mixture. A second component of afirst two-component gel and a second component of the secondtwo-component gel can be combined to form a second mixture. Cells can besuspended within either the first mixture or the second mixture. Whenthe two mixtures are combined, a bioscaffolding including at least someadvantageous characteristics of both gelation systems can be realized.In some embodiments, a bioscaffolding can be formed by mixing at leasttwo different gelation components to form a first mixture. When thefirst mixture is combined with a gelation salt, a bioscaffoldingincluding at least some advantageous characteristics of the individualcomponents can be realized. It should be appreciated that a number ofdifferent combinations of gelation components can be mixed together indifferent ratios to accentuate various advantageous characteristics ofthe individual gelation systems. Furthermore, the concentration of theindividual components, either singly or combined, can influence certaincharacteristics of the bioscaffolding, such as viscosity andencapsulation efficiency.

In some embodiments the bioscaffolding is formed of an alginate andfibrin glue composite material as follows.

EXAMPLE 1

A fibrin glue kit Tisseel™ including modified fibrinogen and thrombinwas obtained from Baxter. Fibrinogen was reconstituted as directed andthen further diluted with water to half of its original concentration.The diluted fibrinogen solution was mixed with a 0.5% alginate-collagensolution in a 1:1 ratio to form a first mixture. hMSC were suspended ina thrombin solution (which contains 40 mM CaCl₂ ) at a concentration of2.96×10⁷ cells/mL. 200 microliters of the first mixture was combinedwith the thrombin in a 1:1 ratio. Encapsulation efficiency of theresulting gel was measured at 91.39±6.78%. The viscosity of thesuspension component was measured at approximately 7 cp.

EXAMPLE 2

A fibrin glue kit Tisseel™ including modified fibrinogen and thrombinwas obtained from Baxter. Fibrinogen was reconstituted as directed andthen further diluted with water to half of its original concentration.The diluted fibrinogen solution was mixed with a 0.5% alginate-collagensolution in a 1:1 ratio to form a first mixture. hMSC were suspended inthe first mixture at a concentration of 5.51×10⁷ cells/mL. The firstmixture was combined with the second mixture comprising thrombin and a2% CaCl₂ solution in a 1:1 ratio. Encapsulation efficiency of theresulting gel was measured at 99.42±0.12%. The viscosity of thesuspension component was measured at approximately 6 cp.

EXAMPLE 3

In one embodiment, a bioscaffolding can be formed by mixing componentsof more than one gelation system. A Tisseel™ kit including a mixture offibrinogen, fibronectin, factor XIII and plasminogen obtained fromBaxter can be dissolved in an aprotinin solution. The combination isthen mixed with gelatin-grafted alginate to form a first mixture. Asecond mixture is formed by combining thrombin (of a fibrin glue system)and calcium chloride. A storage modulus of the combined mixtures mayrange from approximately 0.05 kiloPascals to 150 kiloPascals and morespecifically from approximately 1 kiloPascal to 5 kiloPascals. Aviscosity of the combined mixtures may be approximately 1 cp to 40 cp.In some embodiments, hMSC can be added to an individual component or tothe first or second mixture.

In some embodiments, a bioscaffolding can be formed by mixingalginate-gelatin with sodium-hyaluronate and gelled with calciumchloride. The hyaluronate will be immobilized by chain entanglement andprovide attachment ligands for stem cells bearing the CD44 receptor,e.g., human mesenchymal stem cells. Appropriate formulations can have50% to 99% of a 0.5% to 1.0% solution of alginate-gelatin combined with1% to 50% of a 0.05% to 1% solution of sodium hyaluronate (GenzymeBiosurgery, Mass.). The mixture can be gelled by the addition of anequal volume of a 0.5% to 1.5% of calcium chloride dihydrate.

In another embodiment, a bioscaffolding can be formed by mixingreconstituted lyophilized peptide (with buffer) with alginate and gelledwith calcium chloride. Examples of peptides include self-assembledpeptides, such as RAD 16-II, MAX-1 and EAK16-II. These peptides gel whenexposed to conditions of physiological or greater osmolarity, at neutralpH. However, gelation kinetics are relatively slow, ranging from minutesto hours. Introduction of these peptides as a sole scaffold would resultin an intercalated structure, at best, due to the slow gelation.Addition of alginate can provide rapid gelation kinetics, thuspreventing dissipation of the peptide into tissue.

In some embodiments, a self-assembled peptide (SAP) component can bemixed with a growth factor to form a first mixture. Examples ofself-assembled peptides include RAD 16-II, MAX-1 and EAK16-II. Examplesof growth factors in include, but are not limited to, isoforms ofvasoendothelial growth factor (VEGF), fibroblast growth factor (FGF,e.g. beta-FGF), Del 1, hypoxia inducing factor (HIF 1-alpha), monocytechemoattractant protein (MCP-1), nicotine, platelet derived growthfactor (PDGF), insulin-like growth factor 1 (IGF-1), transforming growthfactor (TGF alpha), hepatocyte growth factor (HGF), estrogens,follistatin, proliferin, prostaglandin E1 and E2, tumor necrosis factor(TNF-alpha), interleukin 8 (I1-8), hematopoietic growth factors,erythropoietin, granulocyte-colony stimulating factors (G-CSF) andplatelet-derived endothelial growth factor (PD-ECGF). The first mixturecan be combined with a component of a two-component gelation system,such as an alginate construct system, a fibrin glue or a polymer system,or any combination thereof. In some embodiments, human mesenchymal stemcells can be added to the system.

In one embodiment, the self-assembled peptide is RAD 16-II and thegrowth factor is PDGF or a derivative thereof and combines to form afirst mixture. It has been shown that PDGF mediates cardiacmicrovascular endothelial cell communication with cardiomyocytes and itis anticipated that an application of PDGF can restore damagedendothelial PDGF-regulated angiogenesis and enhance post-ischemicneovascularization in an infarct region. When combined with RAD 16-II,PDGF binds to RAD 16-II through weak molecular interactions. In someapplications, PDGF remains viable for approximately fourteen days whencombined with RAD 16-II when applied to an infarct region. However, itis anticipated that the slow kinetics of the self-assembled peptide,i.e., minutes to hours, will cause significant leakage, backflow anddissipation before the peptide can form a nanofiber bioscaffolding atthe infarct region.

In some embodiments, the first mixture (comprising RAD 16-II and PDGF)can be combined with any one component of fibrin glue or an alginateconstruct. The rapid kinetics of both fibrin glue and alginateconstructs can counteract the slow kinetics of the SAP-PDGF construct.It is therefore anticipated that leakage and dissipation at the infarctregion can be reduced by combining the SAP-PDGF construct with any onecomponent of fibrin glue or an alginate construct.

The following experimental results using a composite material includingalginate and fibrin glue illustrate how a bioscaffolding may be used toattenuate infarct expansion in a subject having tissues undergoingpost-MI remodeling. Specifically, the results demonstrate that injectionof a composite material such as alginate and fibrin glue, without seededcells and/or growth factors, influence changes in LV geometry and pumpfunction during the post-MI period.

Experiment 1

In particular, in the following experiment, MI was surgically induced inadult pigs. It is noted that pigs are a recognized model forunderstanding post-MI remodeling in humans therefore the treatmentsdisclosed herein are further applicable to humans. It has previouslybeen shown that significant LV remodeling occurs over a one month periodand is accompanied by both regional and global abnormalities in LVperformance. Accordingly, the experiment disclosed herein examined theeffects of forming a bioscaffolding in the MI region in this pig modelwith respect to LV global and regional geometry and function. Theexperiment further examined the relationship of these changes tobiochemical/histological indices of LV myocardial remodeling up to 4weeks post-MI.

The experiment simulated a reasonable post-MI time period by which amyocardial intervention such as injections of components of abioscaffolding could be considered reasonable. The initial wound healingphase and the beginning of mature scar formation occurs approximately 7days post-MI. Wound healing is a process which begins with injury andends with scar formation. It can be separated into three phases:inflammation, proliferation, and remodeling, but these phases overlap tosome degree. In the inflammation phase, fibrin is deposited and acts asa road for future cell infiltration. Neutrophils come in and attack anymicrobes. Macrophages follow and clean up cell debris and releasecytokines to attract and stimulate fibroblasts for the proliferationphase. After 2-3 days, the inflammation phase is complete. In theproliferation phase, fibroblasts come in and release connective tissue,collagen, which supports new blood vessel formation. Fibroblastic andgranulation tissue synthesis takes place during this phase. This phasetakes 1-2 weeks to reach peak fibroblast cell numbers and continues foran additional 2-3 weeks. Contraction also begins during this phase,fibroblasts differentiate into myofibroblasts. This can last for severalweeks, but peaks at 5-10 days post wounding. The remodeling phase ismarked by the deposition and degradation of collagen coming toequilibrium. During this phase, collagen type III is converted tocollagen type I.

It is recognized that interruption or interference of the initial postMI-wound healing response is associated with adverse LV remodeling.Accordingly, in the present study, the composite injections wereperformed at 7 days post-MI in order to avoid the confounding influencessurrounding the acute phase of an MI. It is further contemplated thatthe composite injections may be performed any time during theproliferation phase. Representatively, injections may be made, forexample, 7 to 28 days post wounding, 7 to 14 days post wounding or 7 to10 days post wounding.

For the purposes of assessment of LV regional geometry, radio-opaquemarkers were placed at the initial surgery and allowed for serialassessment infarct expansion. For the purposes of assessing serialchanges in LV global geometry, echocardiography was performed which wasthen complimented by a ventriculographic assessment of LV geometry at 28days post-MI. At 28 days post-MI, a full hemodynamic biochemical andhistological assessment was performed.

Terminology

For the purposes of this experiment, the bioscaffolding componentsinjected into the tissue will include fibrin glue and alginate and willbe referred to as composite injection within the text and Fib-Alg in thetables and figures.

In order to address whether confounding effects of the procedure ofinjection influenced the results over and above that of the compositeinjection, saline injections carried out in identical fashion to thecomposite injections were performed in the protocol. For the purposes ofthis experiment, this group will be termed the saline injection group.

In order to allow for reference comparisons to normal, age matchedcontrols that were not subjected to MI or to injections were included inthe protocol. These measurements served as reference control values forhemodynamics, blood values and biochemistry/histology results.

MI Induction

Permanent coronary ligation was performed in mature pigs (e.g.,Yorkshire, n=21, 25 kg) obtained from Hambone Farms, Orangeburg, S.C. inorder to induce a posterior-lateral MI. On the day of the surgery, thepigs were sedated using 200 mg of benzodiazapam PO obtained fromESI-Elkins-Sinn Inc, NJ, and placed in a custom designed sling. Atransthoracic echocardiogram (e.g., a 3 MHz transducer; Sonos 5500,Agilent Technologies) was performed in order to obtain baselinemeasurements of LV end-diastolic volume, ejection fraction and wallthickness. The pigs were induced with isoflurane (e.g., 3%/1.5 L/minute)and nitrous oxide (e.g., 0.5 L/minute) and intubated. A sterile leftthoracotomy was performed and a catheter connected to an access port(e.g., a 7F obtained from Access Technologies) was placed in thethoracic aorta and the access port placed in a subcutaneous pocket.Next, 4 stainless steel markers (e.g., beads: 1.6 mm outer diameterobtained from VNUS Medical Systems, Sunnyvale, Calif.) were sutured ontothe myocardium centrally located between OM1 and OM2 and 2 cm below thecircumflex artery. The markers were placed for an intermarker distanceof exactly 1 cm (using in-field calibrated instruments) such that themarkers formed a precise quadrilateral array. Two additional markers,placed exactly 1 cm apart, were sutured onto the thoracic wall to serveas an internal calibration. Next, an intravenous bolus of lidocaine(e.g., 1%, 3 mg/kg) was administered and MI induced by direct ligationof the first 2 obtuse marginal branches, OM1 and OM2, at the origin fromthe circumflex coronary artery (e.g., 4.0 Proline). The incision wasclosed in layers. All animals were treated and cared for in accordancewith the National Institute of Health “Guide for the Care and Use ofLaboratory Animals (National Research Council, Washington, 1996).”

Post-MI Measurements and Myocardial Injections

At 7 days post-MI, the pigs were sedated as described in the precedingsection and LV echocardiography performed. Aortic blood pressures andblood samples were collected from the indwelling access port. Using theLV echocardiographic and blood pressure measurements, regional peakcircumferential wall stress was calculated using conventionaltechniques. The blood samples were collected in chilled EDTA tubes,centrifuged and the plasma decanted and stored at −70 degrees C. Theradio-opaque markers were then visualized and digitized usingconventional techniques. Briefly, the fluoroscopic images were recordedfrom orthogonal views and digitized (e.g., 30 frames/s, ATIAll-in-Wonder Radeon, Thornhill, Ontario, Canada) and gated to the ECGin order to identify the end-diastolic frames. The marker coordinatesfrom the corresponding orthogonal frames were merged to determineend-diastolic marker area. End-diastolic marker area was computed fromfive consecutive cardiac cycles.

Following the measurements described above, the pigs were assigned, inalternating fashion, to undergo myocardial injections with the compositematerial or saline. Following assignment to a treatment protocol, thepigs were returned to the operating room and anesthetized withisoflurane as described in the preceding section. An intravenous bolusof lidocaine (e.g., 3 mg/kg) and magnesium chloride (e.g., 30 mg/mL) wasdelivered followed by a continuous infusion of lidocaine (e.g., 30mL/hr). The initial thoracotomy site was reopened, the thoracic spaceirrigated, and the MI region visualized. Using the radio-opaque markersas a frame of reference, a sterile injection guide of plastic laminate(e.g., 0.25 mm) was sutured onto the MI region.

The injection guide encompassed the MI region circumscribed by theObtuse Marginals OM1 and OM2 and extended into the border region of theMI. The injection guide contained a perforated grid with perforations atexactly 0.5 cm intervals and therefore contained 25 perforations over a2×2 cm area. Thus, the placement of this injection guide provided ameans to deliver 25 injections within a precise circumscribed region andpattern.

The composite injection used was made of components of differenttwo-component gel systems. The first component was a mixture of “SealerProtein Concentrate” (Baxter Tisseel™ kit, a mixture of fibrinogen,fibronectin, factor XIII, plasminogen), dissolved in an aprotininsolution, then mixed with gelatin-grafted alginate. The second componentconsisted of a thrombin and calcium chloride solution obtained fromBaxter. The components were prepared under sterile conditions. Thefibrin/alginate mixture and the thrombin/calcium chloride mixture weredrawn into separate 1 mL syringes, loaded onto an injection device suchas a double barreled injection device connected to a 26 gauge needle.This system provided a means to inject 100 μL of both materialssimultaneously with mixing only occurring within the injection needleand at the site of injection. Precisely 200 μL of the injectate wasplaced into the myocardium at an injection depth of 0.5 cm for each ofthe 25 injection sites resulting in a total volume of 5 mL being placedinto the myocardial wall. The composite material polymerized immediatelyat injection and therefore there was no retrograde flow of theinjectate. For the saline injections, the identical syringe system andinjection protocol were followed. Following the injections, the grid wasremoved, and the thoracic space closed and evacuated of air.

LV echocardiography, marker measurements and blood collections were thenrepeated at day 14, 21 and 28 day post-MI (i.e., 7, 14, 21 days postinjection, respectively).

Myocardial Function Measurements at 28 Days Post-MI

Following the final set of serial measurements, the pigs were initiallyanesthetized using 50 μg of intravenous sufentanyl (ESI-Elkins-Sinn Inc,NJ) and intubated. Anesthesia was maintained throughout the procedure bydelivery of 0.5% isoflurane and 60 mg/hr of intravenously administeredmorphine (ESI). An intravenous lactated Ringers solution of 400 mL/hrwas maintained throughout the protocol. This anesthetic protocolresulted in a deep anesthetic plane and stable hemodynamic profile.

A multi-lumened thermodilution catheter (e.g. a 7.5F obtained fromBaxter Healthcare Corp., Irvine, Calif.) was positioned in the pulmonaryartery via the right external jugular vein. An 8F introducer withside-arm was placed in the right carotid for blood pressure measurementsand subsequent placement of the ventriculographic catheter. A sternotomywas performed and a vascular ligature placed around the inferior venacava in order to perform transient caval occlusion.

A previously calibrated microtipped transducer (e.g., a 7.5 F obtainedfrom Millar Instruments Inc, Houston, Tex.) was placed in the LV througha small apical stab wound. The entire posterior-lateral aspect of the LVwas carefully exposed and piezoelectric crystals (e.g., 2 mm obtainedfrom Sonometrics, Ontario) positioned in the central portion of theradio-opaque quadrilateral array. From this crystal array, LV dimensionand wall thickness were recorded at a sampling frequency of 100 Hz anddigitized. A microdialysis probe containing a 4 mm membrane was insertedin the mid-myocardial region between the crystal pairs. Themicrodialysis probe was connected to a precision infusion pump andcontroller system. A flow rate of 2.5 μL/min was established and aniso-osmotic dialysis solution containing a fluorescent substrate for thematrix metalloproteinases (MMPs) was infused at a concentration of 60μM. The dialysate was passed through a micro-fluorescence detector(e.g., FlAlab Instruments, Inc, of Bellevue, Wash.) where the solutionwas subjected to an excitation/emission wavelength of 280/360 nm, andthe output digitized using FlAlab ver. 5. The digitized fluorescentoutput is directly proportional to interstitial myocardial MMP activity.

At the completion of the instrumentation, fluorescent microspheres(e.g., 3×10⁶ obtained from Molecular Probes, Eugene, Oreg.) of specificemission spectra, were injected into the LV. A reference aortic bloodsample was simultaneously withdrawn at a rate of 7 mL/min initiated 5seconds prior to injection and continued for 120 seconds followinginjection. Steady state hemodynamics and microdialysis MMP measurementswere obtained for 60 minutes. Steady state hemodynamics includedsystemic and pulmonary artery pressures, cardiac output, and LVpressures. Following steady-state measurements, LV pre-load was alteredby sequential occlusion and release of the inferior vena cava andisochronal measurements of LV pressure and dimensions recorded. From thedigitized pressure-dimension data, regional myocardial stiffness of theMI region was computed.

LV volumes and ejection fraction were then determined byventriculography. A 6F pigtail catheter was placed into the LV via thecarotid introducer and connected to a pressure-infusion systemcontaining the radio-opaque dye solution. Nonionic contrast material (30cc) was injected into the LV and the opacified image filmed at 60frames/sec in the 30 degree right anterior oblique position.

For the purposes of obtaining reference control values, 5 age and weightmatched pigs were instrumented in the identical fashion and the entireseries of LV myocardial function measurements performed.

Myocardial Sampling and Preparation

Following the final set of measurements, the great vessels werecross-clamped and the heart removed. The LV was quickly separated andplaced in iced saline. A full thickness circumferential ring (e.g., 1cm) was prepared where the sectioning was performed using the centralportion of the marker quadrilateral array as the frame of reference.This section was used for tetrazolium staining and computation of MIsize using planimetry. This myocardial ring was then processed for bloodflow measurements by potassium hydroxide digestion and fluorimetry. Theremaining LV was divided into MI region, border region (defined as the 2cm region surrounding the MI) and the remote region. These myocardialsections were flash frozen for biochemical analysis, or placed informalin for subsequent histological staining.

Myocardial Biochemical and Immunochemical Measurements

LV myocardial samples weighing approximately 0.25 g from each regionwere lyophilized and subjected to hydrochloric acid digestion forhydroxyproline measurements in order to determine total collagencontent. In parallel samples, the myocardium was homogenized andcentrifuged and the supernatant subjected to biochemical measurement ofsoluble collagen using the picrosirius method. Relative MMP-2 and MMP-9levels were determined by substrate zymography. Briefly, LV myocardialextracts (10 μg protein) were subjected to electrophoresis followed bygelatin (e.g., Novex 10% zymogram gel, 0.1% gelatin obtained fromInvitrogen) zymography. The size-fractionated MMP proteolytic regionswere quantified by densitometry using a Gel Pro Analyzer obtained fromMedia Cybernetics.

Plasma MMP Measurements

Plasma measurements of MMP-2 and MMP-9 were performed using an enzymelinked multiplex suspension array obtained from Bio-Rad Laboratories ofHercules, Calif. and flow cytometric detection (Luminex). This approachprovided a sensitivity for MMP-2 at less than 25 pg/mL and for MMP-9 atless than 7 pg/mL with an intra-assay coefficient of variation of lessthan 15%. Using MMP-2 and MMP-9 standards for calibration, a highlinearity for this assay approach was obtained (0.99, p<0.001). Allplasma samples were measured in duplicate.

Myocardial Histology

LV sections of 5 μm were stained with picro-sirius red and the relativecollagen percent area for the MI, border and remote regions weredetermined using computer assisted morphometric methods as describedpreviously. In addition, LV sections were stained with the lectin GSA-B4in order to identify capillary endothelium and compute capillary densityusing computer morphometric methods. Immunohistochemical studies on theparaffin-embedded sections were performed using primary antibodiesdirected against macrophages (MAC-3, CL8943A, Cedarlane, 1:200) andlymphocytes (CD4, CL012A, Cedarlane, 1:200, GEA4023-1, Genex, 1:250).Visualization of the lectin and primary antisera binding sites wasperformed using a 3′,3′-diaminobenzidine-hydrogen peroxide substrateobtained from Vector Labs. Sections were imaged on an invertedmicroscope and the images were digitized using conventional techniques.Negative controls were utilized in all staining protocols and includedsubstitution with nonimmune anti-sera. Routine histological staining wasalso performed in order to examine relative tissue structure in theremote, border and MI regions by hematoxylin and eosin, as well as byAlcian blue counter stained with nuclear fast red.

Data Analysis

All of the data collected in this study were obtained in a blindedfashion and remained coded until the end of the study. The observers forthe echocardiography, marker, and ventriculography measurements wereblinded to the treatment assignments throughout the study protocol.Serial measurements of LV geometry and function were compared using atwo-way analysis of variance (ANOVA) model. Single point measurementswere compared between treatment and control groups using a one-wayANOVA. Following the ANOVA, pair-wise comparisons were performed usingunpaired t-tests corrected for number of comparisons. In addition,comparisons to baseline values and to 7-day post MI values wereperformed as computing the percent change from respective individualvalues and subjecting these computations to a t-test. For thebiochemical and morphometric studies, comparisons to reference controlvalues were performed using a two way ANOVA in which the treatmenteffects were group and region. All statistical analyses were performedusing the STATA® statistical software package obtained from Statacorp ofCollege Station, Tex. Values of p<0.05 were considered to bestatistically significant.

Results Mortality and Final Sample Sizes

All 21 pigs entered into the study survived the initial instrumentationand MI induction. At 24 hours following the initial MI induction, plasmatroponin values were higher than reference control values by over 5-fold(25.6±3.2 U/mL, p<0.05) with no difference between those randomized tothe Fib/Alg group (29.3±5.2 U/mL) and those randomized to the salinegroup (23.2±4.0 U/mL). At 7 days post-MI, of the 11 pigs assigned to thecomposite injection (Fib-Alg) group, 2 pigs developed intraoperativerefractory ventricular fibrillation during the injection procedure. Noneof the 10 pigs assigned to the saline injection group developedrefractory arrhythmogenesis during the 7 day post-MI procedure. Anadditional pig in the Fib-Alg group developed refractory ventriculartachycardia on post-MI day 14. One pig in the saline injection groupdeveloped ventricular tachycardia on post-MI day 28, but wassuccessfully cardioverted for the LV function and hemodynamicmeasurements. Thus, the final sample sizes for this study that completedthe 28 day post-MI protocol were 8 in the Fib-Alg group and 9 in theSaline group.

Serial Measurements

Serial LV echocardiographic measurements are shown in Table 1 below.

TABLE 1 Serial Changes in Echocardiographically-Derived ParametersFollowing Myocardial Infarction (MI): Effects of Saline orFibrin-Alginate Injection at 7 days Post-MI Time Post-MI (days) Baseline7 14 21 28 Heart Rate (bpm) Saline  119 ± 6  117 ± 8  116 ± 4  118 ± 2 129 ± 24 Fib-Alg  130 ± 9  123 ± 4  135 ± 6  123 ± 4  121 ± 7 PosteriorWall thickness at End-systole (cm) Saline 1.10 ± 0.05 0.73 ± 0.08^(#)0.69 ± 0.07^(#) 0.73 ± 0.09^(#) 0.67 ± 0.08^(#) Fib-Alg 1.13 ± 0.04 0.80± 0.06^(#) 1.07 ± 0.11*⁺ 0.90 ± 0.12 0.86 ± 0.11^(#) Septal Wallthickness at End-systole (cm) Saline 1.10 ± 0.03 1.05 ± 0.03 1.09 ± 0.051.18 ± 0.05⁺ 1.23 ± 0.07^(#+) Fib-Alg 1.04 ± 0.03 1.05 ± 0.04 1.10 ±0.03 1.19 ± 0.04^(#+) 1.20 ± 0.04^(#+) End-diastolic Volume (mL) Saline47.5 ± 2.1 55.3 ± 1.9^(#) 59.4 ± 1.5^(#) 69.9 ± 2.2^(#+) 75.6 ± 3.2^(#+)Fib-Alg 45.3 ± 1.0 55.5 ± 1.6^(#) 62.1 ± 2.7^(#) 64.9 ± 2.9^(#+) 71.7 ±3.9^(#+) Ejection Fraction (%) Saline 67.0 ± 0.9 53.2 ± 1.1^(#) 49.8 ±1.6^(#) 47.0 ± 1.9^(#+) 45.6 ± 3.2^(#+) Fib-Alg 68.1 ± 1.0 54.7 ±1.7^(#) 51.2 ± 1.2^(#) 48.2 ± 3.8^(#+) 49.0 ± 2.6^(#) LV Mass/BodyWeight (g/kg) Saline  4.4 ± 0.4  4.2 ± 0.3  3.8 ± 0.3  3.8 ± 0.3  3.5 ±0.2 Fib-Alg  3.7 ± 0.6  3.6 ± 0.5  3.6 ± 0.5  3.1 ± 0.4  3.4 ± 0.5Values presented as Mean ± SEM. Sample Size: Saline: n = 9, Fib-Alg: n =8. ^(#)p < 0.05 vs. Baseline. ⁺p < 0.05 vs. 7 days post-MI. *p < 0.05vs. Saline.

LV systolic posterior wall thickness was reduced and septal wallthickness was increased in a time dependent manner post-MI. However inthe Fib-Alg group, LV posterior wall thickness was similar to baselinevalues, and was comparatively higher than the saline group at 14 dayspost-MI.

LV posterior wall thickness computed as a change from baseline and from7 day post-MI values are shown in FIGS. 2A and 2B, respectively. LVposterior wall thickness fell in both groups, but was higher in theFib-Alg group when compared to the saline group at 14 days post-MI. LVend-diastolic volume increased in a time dependent manner in bothgroups, however, LV end-diastolic volume tended to be lower in theFib-Alg group.

LV end-diastolic volume was computed as a change from individualbaseline or 7 day post-MI values and is shown in FIGS. 3A and 3B,respectively. A trend for a lower LV end-diastolic volume was observedin the Fib-Alg group when compared to respective 7 day values, but thisdid not reach statistical significance. While LV ejection fraction fellin both groups post-MI, LV ejection faction tended to be higher in theFib-Alg group at 28 days post-MI.

The relative changes in LV ejection fraction as a function from baselinevalues and from 7 day post-MI values are shown in FIGS. 4A and 4B,respectively. This analysis revealed that the decline in LV ejectionfraction by 28 days post-MI was less pronounced in the Fib-Alg group.

Serial changes in the radio-opaque markers positioned within the MIregion are shown in FIGS. 5A, 5B and 5C. FIG. 5A illustrates anend-diastolic marker area and FIG. 5B illustrates an end-diastolicmarker area as a function from baseline values. End-diastolic markerarea increased in a time dependent fashion in the saline group, and thisincrease was blunted in the Fib-Alg group. As shown as a function of 7day post-MI values in FIG. 5C, the degree of marker expansion,indicative of infarct expansion, was significantly lower in the Fib-Alggroup when compared to the saline group. Thus, myocardial injection ofFib-Alg at 7 days post-MI reduced the degree of infarct expansion thatprogressively occurs by 28 days post-MI.

Post-MI Day 28 Terminal Studies

At the completion of the 28 day post-MI protocol, additional LVmyocardial measurements of geometry and function were performed. Thesemeasurements were done with full instrumentation and a surgicalprocedure. During the placement of the sonomicrometry crystals, 2 pigsin the Fib/Alg group and 1 pig in the saline group developed refractoryventricular fibrillation. Thus, the hemodynamics on Table 2 below arefor a sample size of 6 and 8 respectively.

TABLE 2 Hemodynamic Parameters at 28 days Following MyocardialInfarction (MI): Effects of Saline or Fibrin-Alginate (Fib-Alg)Injection at 7 days Post-MI Control Saline Fib-Alg Heart Rate (bpm) 106± 6  114 ± 7  135 ± 9^(C ) LV Peak Pressure (mmHg) 113 ± 2  110 ± 2  107± 2  LV End-diastolic Pressure 10 ± 1 10 ± 1 11 ± 1 (mmHg) Peak dP/dt(mmHg/s) 1372 ± 121 1668 ± 212 1814 ± 161 Aortic Systolic Pressure 113 ±2  112 ± 1  107 ± 4  (mmHg) Aortic Diastolic Pressure 80 ± 4 75 ± 3 71 ±4 (mmHg) Mean Aortic Pressure 94 ± 1 93 ± 3 88 ± 4 (mmHg) PA SystolicPressure (mmHg) 27 ± 2 27 ± 1 32 ± 3 PA Diastolic Pressure 17 ± 2 16 ± 121 ± 3 (mmHg) Mean PA Pressure (mmHg) 22 ± 2 21 ± 1 26 ± 4 CardiacOutput (L/min)  3.8 ± 0.6  3.5 ± 0.2  3.8 ± 0.2 Cardiac Index(mL/min/Kg) 109 ± 4  87 ± 4^(C) 98 ± 5 Systemic Vascular Resistance 2047± 180 2168 ± 145 1888 ± 157 (dyne · s · cm⁻⁵) Pulmonary Vascular 322 ±46 333 ± 30 402 ± 65 Resistance (dyne · s · cm⁻⁵) Sample Size 5 8 6Values presented as Mean ± SEM. ^(C) p < 0.05 vs. Control. *p < 0.05 vs.Saline. PA: Pulmonary Artery

Resting ambient heart rate was higher in the Fib-Alg group when comparedto the saline group. Mean arterial pressure was slightly lower in thepost-MI groups, but was not significantly different from controls;indicative of hemodynamic stability. Cardiac output was slightly lowerin the saline group, and when indexed to body weight, was reduced fromreference controls. Cardiac index was unchanged from control values inthe Fib-Alg group.

LV geometry was assessed by ventriculography. LV volumes and ejectionfraction presented as individual data values and mean values for eachgroup are shown in FIGS. 6A and 6B. Similar to the LV echocardiographicmeasurements, as shown in FIG. 6A, LV end-diastolic volume increasedsignificantly in both MI groups, and tended to be lower in the Fib-Alggroup. As shown in FIG. 6B, LV ejection fraction was reduced in both MIgroups, and tended to be higher in the Fib-Alg group. However, theseglobal indices of volume and function remained similar between bothgroups.

Using the combined LV echocardiography and aortic pressure measurements,LV radial wall stress for the remote and MI regions were computed andare shown in FIGS. 7A and 7B, respectively. Regional radial wall stresswas within the MI region in both post-MI groups, but appeared to be thehighest in the saline group.

In-vivo microdialysis was performed in order to measure interstitial MMPactivity within the MI region. The results are shown in FIGS. 8A and 8B.MMP activity tended to be higher in the saline group when compared toreference control values. However, this did not reach statisticalsignificance. In contrast, MMP interstitial activity was lower in theFib-Alg group when compared to control and MI only values.

Myocardial blood flow, normalized to the remote region (region served byLAD) was reduced in the MI region in both the saline and Fib-Alg groupsas illustrated in FIG. 9. Computed MI size based upon tetrazoliumstaining was 26±7% and when normalized by perimeter or by gravimetricmethods, revealed a similar MI size between groups. The results areshown in FIGS. 10A and 10B.

LV regional myocardial stiffness was calculated from the sonomicrometrycrystals placed within the central portion of the MI and constructingregional pressure-dimension relationships with transient cavalocclusion. The results from this analysis are shown in FIG. 11. Regionalmyocardial stiffness was significantly increased within the MI region inboth post-MI groups. The regional stiffness constant was higher in theFib-Alg group, but this did not reach statistical significance (p=0.19).

LV Myocardial Structure

Representative histological sections stained for hematoxylin and eosinare shown in FIG. 12. A greater degree of inflammatory response andapparent neovascularization could be observed within the border and MIregions of the Fib-Alg group. An intense inflammatory response could bereadily appreciated in the MI region of the Fib-Alg group which was morefocally distributed around amorphous densities-likely that of injectedcomposite material.

FIG. 13 illustrates representative photomicrographs for Alcian bluetissue sections which were counter stained with nuclear fast red.Intense blue staining, likely reflective of proteoglycans and otherglycosaminoglycans, could be readily observed within the border and MIregions of both groups.

FIG. 14A illustrates representative histochemical staining forneutrophils using a myeloperoxidase staining approach. The relativeintensity of neutrophil staining increased in the border region andincreased further within the MI region in both groups. However, thedegree of myeloperoxidase appeared increased within both of theseregions in the Fib-Alg group.

Immunohistochemical staining for macrophages is shown in FIG. 14B.Macrophage positive regions were observed within the border and MIregions of both groups, but the number of macrophage positive cellsappeared increased in the Fib-Alg group.

LV myocardial morphometric measurements of percent collagen within eachLV region and placed in comparison to reference control values is shownin FIGS. 15A and 15B. The values for each animal are plotted along withthe mean values for each group. Relative collagen volume fractionincreased within the border and MI regions in both MI groups. However,relative collagen volume fraction was increased in the border region andreduced within the MI region in the Fib-Alg group.

Capillary density as determined by lectin positive vessels wasdetermined in full thickness LV sections and the results are summarizedin FIG. 16. Capillary density within the MI region was reduced in bothpost-MI groups compared to reference control values. In the salinegroup, capillary density was reduced within the border and MI regionswhen compared to respective remote region values. In marked contrast,capillary density was similar to controls within the border region andnot different from respective remote regions in the Fib-Alg group.

LV Myocardial Biochemistry

Soluble collagen, that is collagen that could be extracted by a bufferedsalt solution and homogenization, was increased within the MI regions ofboth groups as shown in FIG. 17A. This is reflective of newlysynthesized and incomplete collagen cross-linking, which would be morevulnerable to degradation. However, soluble collagen content wasdecreased within the MI region in the Fib-Alg group, indicative of amore highly cross-linked fibrillar collagen within this region. Totalcollagen content, as determined by hydroxyproline measurements of aciddigested myocardial samples was increased in a gradual fashion from theremote to MI regions as illustrated in FIG. 17B. These results areconsistent with a post-MI fibrotic response, with no significantdifferences between MI groups.

LV MMP profiles for MMP-9 and MMP-2, as determined by zymography, areshown in FIGS. 18A and 18B, respectively. MMP-9 levels were increasedsignificantly within the MI region in both groups. While the relativelevels of MMP-9 appeared higher in the Fib-Alg group, these levels didnot reach statistical significance. Thus, the robust inflammatoryresponse observed by histological analysis was not associated with asignificant increase in relative MMP-9 levels, a primary proteolyticproduct of neutrophils. Relative MMP-2 levels were significantlyincreased within the MI regions of both MI groups, but weresignificantly and substantially lower in the Fib-Alg group when comparedto respective saline values. This was most notable for the lowermolecular weight form of MMP-2 (68 kDa) which reflects the active formof MMP-2. LV MMP-2 values and MMP-2 values normalized to a control areshown in FIGS. 19A and 19B.

Plasma MMP Profiles

Despite multiple dilution series, plasma MMP-9 levels were notdetectable within the plasma samples collected in the post-MI period.Plasma levels for MMP-2 were higher than control values at all post-MItime points in the saline group. However, in the Fib-Alg group, plasmaMMP-2 levels were not different from controls after 7 days post-MI. Inaddition, plasma MMP-2 levels were reduced from saline values in theFib-Alg groups at several post-MI time points.

SUMMARY

The key findings of this experiment can be summarized into 3 broadareas: (1) LV function and hemodynamics, (2) myocardial structure, and(3) myocardial biochemistry.

LV Function and Hemodynamics

The main findings in this area was that the Fib-Alg injection into theMI region caused a time dependent increase in posterior LV wallthickness when compared to reference vehicle MI values. In the Fib-Alggroup, the rate of MI expansion was significantly attenuated and globalindices of LV geometry and function appeared to be improved with Fib-Alginjection. Specifically, the relative fall in LV fractional shorteningwas reduced and a measure of LV pump function, cardiac index wasincreased. Likely mechanisms for the significant attenuation in theinfarct expansion process with Fib-Alg are improved preservation of thefibrillar collagen matrix as discussed in a subsequent section. Thebasis for the improvements in LV pump function were likely due tochanges in LV load as regional wall stress patterns and systemicvascular resistance tended to be lower in the Fib-Alg group. All ofthese favorable effects on local and global LV geometry were not due todifferences in initial MI sizes or blood flow patterns.

LV Myocardial Structure

One of the more prominent features from the imaging studies was theincreased LV posterior wall thickness following Fib-Alg injection. Atthe histological level, a clear and robust inflammatory response wasobserved by both histochemical and immunohistochemical methods.Specifically, increased neutrophil infiltrate within the MI region wasobserved at one month post-MI in with Fib-Alg injection and this wasassociated with a higher number of macrophages. Interestingly, however,this was not associated with increased proteolytic activity as assessedby in-vivo microdialysis. In fact, MMP proteolytic activity was reducedwithin the MI region in the Fib-Alg group. Total soluble collagen withinthe MI region was reduced with Fib-Alg injection whereas totalbiochemical content of collagen was similar between the MI groups. Thissuggest that greater cross-linking of collagen occurred in the Fib-Alggroup which would improve infarct stiffening and favor improvedtethering of the border region. Indeed, relative myocardial stiffnessappeared higher in the Fib-Alg group and relative percent collagen bymorphometry was increased within the border region in the Fib-Alg group.Thus, the increased inflammatory response observed in the Fib-Alg groupdid not appear to cause increased collagen degradation, but ratherfacilitated a maturation of collagen and scar formation within the MIand border regions.

LV Myocardial Biochemistry

Relative levels of MMP-9 were increased to a similar fashion in the MIgroups. Again, this is a somewhat surprising finding due to the factthat a greater inflammatory response was observed in the Fib-Alg group.Moreover, myocardial MMP-2 levels, a ubiquitous MMP found in themyocardium and activated during myocardial remodeling, was reduced inthe Fib-Alg MI region. Taken together, this would result in a netreduction in proteolytic activity consistent with the in-vivomicrodialysis measurements. Additional confirmation of the relativereduction in MMP-2 levels in the Fib-Alg group is the relative reductionin circulating MMP-2 levels in the post-MI period.

Overall Conclusion

The injection of a Fib-Alg composite material in the post-MI periodattenuated infarct expansion and afforded favorable effects on regionaland global LV function. The mechanisms by which the Fib-Alg injectionmodified the acceleration of infarct expansion was by altering thecellular infiltrate and collagen composition within the MI and borderregions which favored mature scar formation.

Delivery Systems

Devices which can be used to deliver modified or combined components ofthe gelation systems include, but are not limited to, dual-needleleft-ventricle injection devices, dual-needle transvascular wallinjection devices and dual syringes. Methods of access to use theminimally invasive (i.e., percutaneous or endoscopic) injection devicesinclude access via the femoral artery or the sub-xiphoid. “Xiphoid” or“xiphoid process” is a pointed cartilage attached to the lower end ofthe breastbone or sternum, the smallest and lowest division of thesternum. Both methods are known by those skilled in the art.

FIG. 20 illustrates an embodiment of a dual syringe device which can beused to deliver the compositions of the present invention. Dual syringe400 can include first barrel 410 and second barrel 420 adjacent to oneanother and connected at a proximal end 455, distal end 460 and middleregion 465 by plates 440, 445 and 450, respectively. In someembodiments, barrels 410 and 420 can be connected by less than threeplates. Each barrel 410 and 420 includes plunger 415 and plunger 425,respectively. Barrels 410 and 420 can terminate at a distal end intoneedles 430 and 435, respectively, for extruding a substance. In someembodiments, barrels 410 and 420 can terminate into cannula protrusionsfor extruding a substance. Barrels 410 and 420 should be in close enoughproximity to each other such that the substances in each respectivebarrel are capable of mixing with one another to form a bioscaffoldingin the treatment area, e.g., a post-infarct myocardial region. Dualsyringe 400 can be constructed of any metal or plastic which isminimally reactive or completely unreactive with the formulationsdescribed in the present invention. In some embodiments, dual syringe400 includes a pre-mixing chamber attached to distal end 465.

In some applications, first barrel 410 can include a first mixture of amodified two-component gelation system and second barrel 420 can includea second mixture of a modified two-component gelation system accordingto any of the embodiments described previously. A therapeutic amount ofthe resulting gel is between about 25 μL to about 200 μL, preferablyabout 50 μL. Dual syringe 400 can be used during, for example, an openchest surgical procedure.

FIGS. 21A-21B illustrate an embodiment of a dual-needle injection devicewhich can be used to deliver the compositions of the present invention.Delivery assembly 500 includes lumen 510 which may house deliverylumens, guidewire lumens and/or other lumens. Lumen 510, in thisexample, extends between distal portion 505 and proximal end 515 ofdelivery assembly 500.

In one embodiment, delivery assembly 500 includes first needle 520movably disposed within delivery lumen 530. Delivery lumen 530 is, forexample, a polymer tubing of a suitable material (e.g., polyamides,polyolefins, polyurethanes, etc.). First needle 520 is, for example, astainless steel hypotube that extends a length of the delivery assembly.First needle 520 includes a lumen with an inside diameter of, forexample, 0.08 inches (0.20 centimeters). In one example for aretractable needle catheter, first needle 520 has a needle length on theorder of about 40 inches (about 1.6 meters) from distal portion 505 toproximal portion 515. Lumen 510 also includes auxiliary lumen 540extending, in this example, co-linearly along the length of the catheter(from a distal portion 505 to proximal portion 515). Auxiliary lumen 540is, for example, a polymer tubing of a suitable material (e.g.,polyamides, polyolefins, polyurethanes, etc.). At distal portion 505,auxiliary lumen 540 is terminated at a delivery end of second needle 550and co-linearly aligned with a delivery end of needle 520. Auxiliarylumen 540 may be terminated to a delivery end of second needle 550 witha radiation-curable adhesive, such as an ultraviolet curable adhesive.Second needle 550 is, for example, a stainless steel hypotube that isjoined co-linearly to the end of main needle 520 by, for example, solder(illustrated as joint 555). Second needle 550 has a length on the orderof about 0.08 inches (0.20 centimeters). FIG. 21B shows across-sectional front view through line A-A′ of delivery assembly 500.FIG. 21B shows main needle 520 and second needle 550 in a co-linearalignment.

Referring to FIG. 21A, at proximal portion 515, auxiliary lumen 540 isterminated to auxiliary side arm 460. Auxiliary side arm 560 includes aportion extending co-linearly with main needle 520. Auxiliary side arm560 is, for example, a stainless steel hypotube material that may besoldered to main needle 520 (illustrated as joint 565). Auxiliary sidearm 560 has a co-linear length on the order of about, in one example,1.2 inches (3 centimeters).

The proximal end of main needle 520 includes adaptor 570 foraccommodating a substance delivery device. Adaptor 570 is, for example,a molded female luer housing. Similarly, a proximal end of auxiliaryside arm 560 includes adaptor 580 to accommodate a substance deliverydevice (e.g., a female luer housing).

The design configuration described above with respect to FIGS. 21A-21Bis suitable for introducing modified two-component gel compositions ofthe present invention. For example, a gel may be formed by a combination(mixing, contact, etc.) of a first mixture of a modified two-componentgelation system and a second mixture of a modified two-componentgelation system. Representatively, a first mixture may be introduced bya one cubic centimeters syringe at adaptor 570 through main needle 520.At the same time or shortly before or after, a second mixture may beintroduced with a one cubic centimeter syringe at adaptor 580. When thefirst and second components combine at the exit of delivery assembly 500(at an infarct region), the materials combine (mix, contact) to form abioerodable gel.

FIGS. 22A-22C illustrate an alternative embodiment of a dual-needleinjection device which can be used to deliver two-component gelcompositions of the present invention. In general, the catheter assembly600 provides a system for delivering substances, such as modifiedtwo-component gel compositions, to or through a desired area of a bloodvessel (a physiological lumen) or tissue in order to treat a myocardialinfarct region. The catheter assembly 600 is similar to the catheterassembly 600 described in commonly-owned, U.S. Pat. No. 6,554,801,titled “Directional Needle Injection Drug Delivery Device”, which isincorporated herein by reference.

In one embodiment, catheter assembly 600 is defined by elongatedcatheter body 650 having proximal portion 620 and distal portion 610.Guidewire cannula 670 is formed within catheter body (from proximalportion 610 to distal portion 620) for allowing catheter assembly 600 tobe fed and maneuvered over guidewire 680. Balloon 630 is incorporated atdistal portion 610 of catheter assembly 600 and is in fluidcommunication with inflation cannula 660 of catheter assembly 600.

Balloon 630 can be formed from balloon wall or membrane 635 which isselectively inflatable to dilate from a collapsed configuration to adesired and controlled expanded configuration. Balloon 630 can beselectively dilated (inflated) by supplying a fluid into inflationcannula 660 at a predetermined rate of pressure through inflation port665 (located at proximal end 620). Balloon wall 635 is selectivelydeflatable, after inflation, to return to the collapsed configuration ora deflated profile. Balloon 630 may be dilated (inflated) by theintroduction of a liquid into inflation cannula 660. Liquids containingtreatment and/or diagnostic agents may also be used to inflate balloon630. In one embodiment, balloon 630 may be made of a material that ispermeable to such treatment and/or diagnostic liquids. To inflateballoon 630, the fluid can be supplied into inflation cannula 660 at apredetermined pressure, for example, between about one and 20atmospheres. The specific pressure depends on various factors, such asthe thickness of balloon wall 635, the material from which balloon wall635 is made, the type of substance employed and the flow-rate that isdesired.

Catheter assembly 600 also includes at least two substance deliveryassemblies 605 a and 605 b (not shown; see FIGS. 22B-22C) for injectinga substance into a myocardial infarct region. In one embodiment,substance delivery assembly 605 a includes needle 615 a movably disposedwithin hollow delivery lumen 625 a. Delivery assembly 605 b includesneedle 615 b movably disposed within hollow delivery lumen 625 b (notshown; see FIGS. 6B-6C). Delivery lumen 625 a and delivery lumen 625 beach extend between distal portion 610 and proximal portion 620.Delivery lumen 625 a and delivery lumen 625 b can be made from anysuitable material, such as polymers and copolymers of polyamides,polyolefins, polyurethanes and the like. Access to the proximal end ofdelivery lumen 625 a or delivery lumen 625 b for insertion of needle 615a or 615 b, respectively is provided through hub 635 (located atproximal end 620). Delivery lumens 625 a and 625 b may be used todeliver first and second mixtures of a modified two-component gelcomposition to a post-myocardial infarct region.

FIG. 22B shows a cross-section of catheter assembly 600 through lineA-A′ of FIG. 22A (at distal portion 610). FIG. 22C shows a cross-sectionof catheter assembly 600 through line B-B′ of FIG. 22A. In someembodiments, delivery assemblies 605 a and 605 b are adjacent to eachother. The proximity of delivery assemblies 605 a and 605 b allows eachmixture of the modified two-component gelation system to rapidly gelwhen delivered to a treatment site, such as a post-myocardial infarctregion.

From the foregoing detailed description, it will be evident that thereare a number of changes, adaptations and modifications of the presentinvention which come within the province of those skilled in the part.The scope of the invention includes any combination of the elements fromthe different species and embodiments disclosed herein, as well assubassemblies, assemblies and methods thereof. However, it is intendedthat all such variations not departing from the spirit of the inventionbe considered as within the scope thereof.

What is claimed is:
 1. A method comprising: forming a bioscaffoldingcomprising a first two-component gelation system and a secondtwo-component gelation system within a post-myocardial infarct regionsufficient to cause attenuation of a rate of myocardial infarctexpansion.
 2. The method of claim 1, wherein the bioscaffolding isformed during a proliferation phase occurring after a myocardialinfarction.
 3. The method of claim 1, wherein the bioscaffolding isformed seven days or more after occurrence of a myocardial infarction.4. The method of claim 1, further comprising: forming a bioscaffoldingwithin a border region of the post-myocardial infarct region.
 5. Themethod of claim 1, wherein (a) a first component of the firsttwo-component gelation system comprises gelatin grafted alginate and afirst component of the second two-component gelation system is afibrinogen solution and (b) a second component of the firsttwo-component gelation system is thrombin and a second component of thesecond two-component gelation system is calcium chloride.
 6. The methodof claim 1, wherein a storage modulus of the bioscaffolding is from 0.05kiloPascals to 150 kiloPascals.
 7. The method of claim 1, wherein astorage modulus of the bioscaffolding is from 1 kiloPascal to 5kiloPascals.
 8. A method comprising: forming a bioscaffolding within apost-myocardial infarct region sufficient to cause a time dependentincrease in posterior left ventricular wall thickness.
 9. The method ofclaim 8, wherein forming the bioscaffolding comprises deliveringcomponents of a first two-component gelation system and components of asecond two-component gelation system to the post-myocardial infarctregion within a predetermined time period after occurrence of amyocardial infarction.
 10. The method of claim 9, wherein thepredetermined time period is seven days or more.
 11. The method of claim8, further comprising: forming a bioscaffolding within a border regionof the post-myocardial infarct region.
 12. The method of claim 9,wherein (a) a first component of the first two-component gelation systemcomprises gelatin grafted alginate and a first component of the secondtwo-component gelation system is a fibrinogen solution and (b) a secondcomponent of the first two-component gelation system is thrombin and asecond component of the second two-component gelation system is calciumchloride.
 13. A method comprising: forming a bioscaffolding within apost-myocardial infarct region sufficient to cause a maturation ofmyocardial scar formation.
 14. The method of claim 13, wherein forming abioscaffolding decreases matrix metalloproteinase activity.
 15. Themethod of claim 13, wherein forming a bioscaffolding increases collagentype I.
 16. The method of claim 13, wherein forming a bioscaffoldingincreases capillary density.
 17. The method of claim 13, wherein forminga bioscaffolding increases fibroblast infiltration.
 18. The method ofclaim 13, wherein forming a bioscaffolding comprises forming thebioscaffolding during a proliferation phase of wound healing.
 19. Themethod of claim 13, wherein forming a bioscaffolding comprises formingthe bioscaffolding seven days or more after occurrence of a myocardialinfarction.
 20. The method of claim 13, further comprising: forming abioscaffolding within a border region of the post-myocardial infarctregion.
 21. The method of claim 13, wherein the bioscaffolding comprisesa first two-component gelation system and a second two-componentgelation system wherein (a) a first component of the first two-componentgelation system comprises gelatin grafted alginate and a first componentof the second two-component gelation system is a fibrinogen solution and(b) a second component of the first two-component gelation system isthrombin and a second component of the second two-component gelationsystem is calcium chloride.
 22. A composition of matter comprising: apost-myocardial infarct tissue comprising a bioscaffolding that resistsexpansion resulting in attenuation of a rate of myocardial infarctexpansion of the tissue.
 23. The composition of matter of claim 22,wherein the bioscaffolding comprises a first two-component gelationsystem and a second two-component gelation system.
 24. The compositionof matter of claim 23, wherein (a) a first component of the firsttwo-component gelation system comprises gelatin grafted alginate and afirst component of the second two-component gelation system is afibrinogen solution and (b) a second component of the firsttwo-component gelation system is thrombin and a second component of thesecond two-component gelation system is calcium chloride.